Organophosphorus degrading enzyme based multifunctional catalyst and preparation method and use thereof

ABSTRACT

The present disclosure provides a method for preparing an organophosphorus degrading enzyme based multifunctional catalyst and an organophosphorus degrading enzyme based multifunctional catalyst and use thereof. In the present disclosure, the preparation method includes: directly adding a composite yolk-shell-structured nanomaterial into a crude enzyme solution of organophosphorus degrading enzyme with an affinity tag, and mixing, to obtain a mixture, and then subjecting the mixture to a separation, to obtain an organophosphorus degrading enzyme based multifunctional catalyst. According to the present disclosure, the method for preparing an organophosphorus degrading enzyme based multifunctional catalyst is simple in operation, and has a low cost; the multifunctional catalyst prepared by the same has low requirement for the purity of enzyme, support of which could be directionally binded with enzyme, and could be used for detecting an organophosphorus pesticide, and also for a cascade degradation of an organophosphorus pesticide. The final product p-aminophenol has important application value.

TECHNICAL FIELD

The present disclosure relates to the technical field of the preparationof biocatalyst, and in particular to a method for preparing anorganophosphorus degrading enzyme based multifunctional catalyst.Furthermore, the present disclosure relates to the organophosphorusdegrading enzyme based multifunctional catalyst prepared by the methodand use of the same in the detection and degradation of anorganophosphorus pesticide.

BACKGROUND

Organophosphorus pesticide is a pesticide widely used in agricultureinsecticide, and it comprises a large amount of organophosphoruscompound. However, the organophosphorus compound is one of currentlyknown substances that has a high toxicity. A wide use of theorganophosphorus pesticide has caused great pollution to theenvironment, and serious hazard to human health; thus, the detection anddegradation thereof is very important.

In recent years, great progress has been made in the research ofbiodegradation of organophosphorus pesticide by using biotechnology.Compared with the potential drawbacks of traditional methods, biologicalmethods have been more attractive. The biological method has a lessdestruction and a lower cost. The catalyst used, however, exhibits poorstability, and thus could not be recycled; the purification steps arecomplex, and the degradation is incomplete, which limits theapplications of the biological method.

It is well known that the immobilization of enzyme is an effectivemethod to solve the problems of the instability and non-recyclability ofenzyme catalyst, but all traditional immobilization methods need apurification step of enzyme prior to the immobilization of enzyme. Inorder to solve the complex enzyme purification step, the enzyme genesand the histidine affinity tag genes are always recombined and thenpurified. Thus, in recent years, people pay more and more attention tothe protein separation and purification system based on nanoparticlesfunctionalized by immobilized metal affinity chromatography (IMAC).However, the steps of such method are still tedious, and in the use ofchelating agent, when the metal ions of metal enzyme active center mayinteract with chelating agent, the metal enzyme activity would benegatively affected (such as organophosphate degrading enzyme).

SUMMARY

In view of this, the present disclosure is to provide a method forpreparing an organophosphorus degrading enzyme based multifunctionalcatalyst, and the organophosphorus degrading enzyme basedmultifunctional catalyst prepared by the same could overcome theproblems of the existing organophosphorus degrading enzyme: poorstability and incomplete degradation in terms of the final product whenused to degrade the organophosphorus pesticide.

In order to achieve the above object, the technical solutions of thepresent disclosure are provided as follows:

A method for preparing an organophosphorus degrading enzyme basedmultifunctional catalyst, comprising,

directly adding a composite yolk-shell-structured nanomaterial into acrude enzyme solution of an organophosphorus degrading enzyme with anaffinity tag, and mixing, to obtain a mixture, and subjecting themixture to a separation, to obtain the organophosphorus degrading enzymebased multifunctional catalyst.

In some embodiments, the composite yolk-shell-structured nanomaterial isCo/C@SiO₂@Ni/C.

In some embodiments, the affinity tag is selected from the groupconsisting of histidine tag, cysteine tag, and tryptophan tag.

In some embodiments, the organophosphorus degrading enzyme is coded by agene sequence obtained from soil pseudomonas, flavobacterium, oragrobacterium radioactive.

In some embodiments, the mixing is performed by using a shaking table orstirring for 0.5-6 hours; subjecting the mixture to a separation isperformed by a centrifugation or filtration.

Compared with the prior art, the present disclosure has the followingbeneficial effects:

In the method for preparing an organophosphorus degrading enzyme basedmultifunctional catalyst according to the present disclosure, thecomposite yolk-shell-structured nanomaterial which is rich in transitionmetal ions in itself is used to further purify and immobilize theorganophosphorus degrading enzyme with an affinity tag, and it wouldeffectively solve the drawbacks of the poor stability, inability torecycle, and complex purification during the enzyme catalysis. Themethod as described above is simple in operation, has a lowerrequirement for the purity of enzyme, thus there is no need to carry outcostly separation and purification of enzyme, and the support could bedirectionally binded with enzyme. The prepared organophosphorusdegrading enzyme based multifunctional catalyst could be used fordetecting the organophosphorus pesticide, and also for a cascadedegradation of the organophosphorus pesticide.

Furthermore, the present disclosure provides an organophosphorusdegrading enzyme based multifunctional catalyst prepared by the methodfor preparing an organophosphorus degrading enzyme based multifunctionalcatalyst as described above.

In addition, the present disclosure also provides use of theorganophosphorus degrading enzyme based multifunctional catalyst fordetecting or degrading an organophosphorus pesticide.

In some embodiments, detecting the organophosphorus pesticide isperformed by an optical detection method.

In some embodiments, the organophosphorus degrading enzyme basedmultifunctional catalyst is used in accompany with a hydrogen donor todegrade the organophosphorus pesticide.

In some embodiments, the hydrogen donor is sodium borohydride.

The organophosphorus degrading enzyme based multifunctional catalystcould be used for detecting or degrading an organophosphorus pesticide.The final product obtained after the degradation is non-toxic, and havea wide range of applications and good application effects.

BRIEFT DESCRIPTION OF THE DRAWINGS

The accompanying drawings which constitute part of the presentdisclosure are used to provide a further understanding of the presentdisclosure. The schematic embodiments of the present disclosure and theillustrations thereof are used to explain the present disclosure, and donot constitute an undue limitation of the present disclosure. In theaccompanying drawings:

FIG. 1 shows a scanning electron micrograph of Co/C@SiO₂@Ni/C of Example1 of the present disclosure;

FIG. 2 shows a transmission electron micrograph of Co/C@SiO₂@Ni/C ofExample 1 of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

It is noted that the embodiments in the present disclosure and thecharacteristics in the embodiments may be combined with each other ifnot conflict. At the same time, the specific conditions not specified inthe present disclosure, may be conventional conditions or thoserecommend by the manufacturers of equipments used. The reagents orinstruments used that are not specified in manufacturers, may beconventional and commercially available products. For the technicalmeans or techniques involved, if the conditions are not specified, itmay mean that the existing means in the field thereof could be used.

The present disclosure will be further described in detail below inconjunction with embodiments and accompanying drawings.

This embodiment involves a method for preparing an organophosphorusdegrading enzyme based multifunctional catalyst, and in the method, acomposite yolk-shell-structured nanomaterial is used as a support, andan organophosphorus degrading enzyme with an affinity tag is used as atarget enzyme. The support is directly added into a crude enzymesolution of a target enzyme, and they are adequately mixed for reacting,to obtain a mixture, and the mixture is subjected to a separation, toobtain the organophosphorus degrading enzyme based multifunctionalcatalyst.

In this embodiment, the composite yolk-shell-structured nanomaterial isa mesoporous material with a clear core-shell structure, which is formedfrom a self-assembly of an organic ligand containing oxygen and nitrogen(mostly aromatic polyacids and polybases) and a transition metal ion,after being covered with silica, coated with dopamine and a transitionmetal ion, and then being calcined. The composite yolk-shell-structurednanomaterial has the advantages of low density, large surface area, goodchemical and mechanical stability, and high permeability for charge andmolecule transport. According to the present disclosure, the compositeyolk-shell-structured nanomaterial is used as a support to prepare theorganophosphorus degrading enzyme based multifunctional catalyst.

The affinity tag is a sequence of amino acid that has a high affinityfor a particular biological or chemical ligand. The fusion of theaffinity tag with a protein is not only convenient for the detection andpurification of the fused protein, but also affects the physical andchemical properties of the target protein. The fusion, however, isgenerally required not to produce influences, otherwise which wouldnegatively affect the activity of the enzyme. According to the presentdisclosure, the organophosphate degrading enzyme with an affinity tag isselectively binded with the support, through the interaction between thetransition metal ions (Cu²⁺, Ni²⁺, Co²⁺, Zn²⁺, etc.) on the support ofthe composite yolk-shell-structured nanomaterial with existing aminoacid residues (e.g., imidazolyl, sulfydryl and indolyl group(s) ofhistidine, cysteine and tryptophan).

The organophosphate degrading enzyme is a kind of protein that cancatalyze P—O bond, P—F bond and P—S bond in the organophosphorussubstance to crack, and is featured by high catalytic efficiency and awide range of substrate when used for the degradation of theorganophosphorus pesticide, and thus could be used to catalyze thedegradation of many kinds of organic phosphorus compound (such asparathion-methyl, paraoxon, parathion, coumaphos, diazinon andisoflurophate). Thus, in order to protect the environment and humanhealth, reducing the residues of the organophosphorus compound innature, degrading the organophosphorus compound by usingorganophosphorus degrading enzyme has a great prospect. According to thepresent disclosure, the organophosphorus degrading enzyme is selected tobind with the support to prepare an immobilized enzyme, which is usedfor the degradation of the organophosphorus compound in the environment.

Although the organophosphorus degrading enzymes could be used to degradethe organophosphorus compound, only degrading them into p-nitrophenol.Although the toxicity of p-nitrophenol is many times lower than that ofthe organophosphorus compound, it is still a toxic substance that couldcause environmental pollution, thus, it could not completely solve thepollution problem of the organophosphorus compound to the environment.In order to better restore the ecosystem damaged by the organophosphoruspesticide, on the basis of the organophosphorus degrading enzyme, theimmobilization thereof is achieved by using the compositeyolk-shell-structured nanomaterial, and the obtained catalyst is used tocatalyze the further degradation of p-nitrophenol to generatep-aminophenol, in the presence of a hydrogen donor. Moreover,p-aminophenol is an important pharmaceutical intermediate, and thus thecatalyst could not only enable the complete degradation oforganophosphorus compound, but also provide a wide range of applicationvalue.

Crude enzyme solution refers to a mixed solution containing the targetenzyme obtained by removing macromolecular substances, such as cellwalls or organelles, from a microorganism, after microbial fermentation.The existing process for preparing of a biocatalyst has a highrequirement for the purity of the target enzyme, and thus it is neededto subject the crude enzyme solution obtained after microbialfermentation to complex separation and purification process(es), whichleads to a high cost. According to the present disclosure, directlycombining the support with the crude enzyme solution could omit theseparation and purification step of the crude enzyme solution, therebygreatly reducing the production cost.

According to the present disclosure, in the method, a compositeyolk-shell-structured nanomaterial is used as a support, and anorganophosphorus degrading enzyme with an affinity tag is used as atarget enzyme. The support is directly added into a crude enzymesolution containing the target enzyme, and they are adequately mixed forreacting, to obtain a mixture, and the mixture is subjected to aseparation, to obtain the organophosphorus degrading enzyme basedmultifunctional catalyst.

As the organic ligand and metal ions in the compositeyolk-shell-structured nanomaterial could be selected, thus differenttypes of yolk-shell-structured nanomaterial could be synthesized bycombining different types of organic ligands and different transitionmetal elements.

According to the present disclosure, the material with betterperformance could be selected as the support by limiting the type of thecomposite yolk-shell-structured nanomaterial, in order to make theprepared organophosphorus degrading enzyme based multifunctionalcatalyst exhibit higher enzyme utilization rate, and better enzymeactivity. By further selecting the yolk-shell-structured nanomaterial,the organophosphorus degrading enzyme utilization rate and the enzymeactivity of the organophosphorus degrading enzyme based multifunctionalcatalyst could be further increased.

In some embodiments, a preferred composite yolk-shell-structurednanomaterial is generally prepared by a method comprising the followingsteps:

A 2-methyl imidazole solution and a Co(NO₃)₂.6H₂O solution are mixed,fully shook and sonicated for 10 min, stirred at room temperature for 6h, and the resulting mixture is subjected to a separation by acentrifugation, to obtain a precipitate. The precipitate (i.e. ZIF67) iswashed in sequence with ultrapure water and ethanol 3 times each, andthe washed precipitate was ultrasonically dispersed into anhydrousethanol. The 2-methyl imidazole solution is added therein, and they arefully shook and sonicated for 5 min to be uniform. Then acetyltrimethylammonium chloride (5 wt %) solution is added therein, andthey are stirred for 5 min at room temperature. Then, ethylorthosilicate (TEOS) is slowly added dropwise into the mixed solutionabove and they are stirred at room temperature for 1.5 h. The resultingmixture is subjected to a separation by a centrifugation, to obtain aprecipitate. The precipitate (ZIF67@SiO₂) is washed with ultrapure water3 times and washed with ethanol 3 times. The washed precipitate isultrasonically dispersed in ethanol solution again and mixed with Trissolution, to obtain a mixed solution. Then dopamine and NiCl₂.6H₂O areadded into the mixed solution in sequence, and after dopamine andNiCl₂.6H₂O being dissolved completely, the resulting mixture are stirredat room temperature for 15 h. The mixture obtained after stirring issubjected to a separation by a centrifugation (9000 rpm, 5 min), toobtain a precipitate. The precipitate is washed with ultrapure water andethanol several times, and the washed precipitate is dried, to obtain aprecursor material. The precursor material is placed in a quartzcrucible and calcined at 500° C. for 2 h, with a heating rate of 2°C.·min⁻¹, under the protection of nitrogen, to obtain the compositeyolk-shell-structured nanomaterial Co/C@SiO₂@Ni/C.

In some embodiments, a preferred organophosphorus degrading enzyme basedmultifunctional catalyst is generally prepared by a method comprisingthe following steps.

The composite yolk-shell-structured nanomaterial is added into theTris-HCl buffer solution, and they are sonicated for 2 mM to beuniformly dispersed, and the uniformly dispersed compositeyolk-shell-structured nanomaterial is added into a crude enzymesolution, and they are evenly mixed, shook on a shaking table at roomtemperature for 3 h. The resulting mixture is subjected to acentrifugation, to obtain a precipitate. The precipitate is washed withimidazole buffer solution (40 mM) three times, to obtain a washedprecipitate, i.e. the organophosphorus degrading enzyme basedmultifunctional catalyst OpdA@Co/Ad@Ni/C.

In some embodiments, the affinity tag includes histidine tag (His-Tag),cysteine tag (Cys Tag) and tryptophan tag (Trp-Tag), and is preferablyhistidine tag, mainly because histidine tag has a small molecular weightand generally does not affect the target protein. By further selectingthe type of the affinity tag, the target enzyme with an affinity tagcould be binded with the support more firmly, so as to improve theenzyme utilization rate and the enzyme activity of the organophosphorusdegrading enzyme based multifunctional catalyst. However, in addition tothe affinity tags described above, other affinity tags that could bebinded with transition metal ions known to those skilled in the art maybe used.

In some embodiments, the gene sequence that codes the organophosphorusdegrading enzyme in the organophosphorus degrading enzyme with anaffinity tag is obtained from pseudomonas agrobacterium, flavobacterium,or radioactive agrobacterium, and preferably is obtained from the genesequence that codes organophosphorus degrading enzyme in radioactiveagrobacterium. By the existing genetic engineering method, the affinitytags is introduced into the N-terminal of the gene that codesorganophosphorus degrading enzyme, to obtain the desired gene sequence.Then the desired gene sequence is transformed in a transforming plasmidand expressed in a expressing bacteria, to obtain the organophosphorusdegrading enzyme with an affinity tags. There are types oforganophosphorus degrading enzyme, and different types oforganophosphorus degrading enzymes exhibit different catalyticefficiency and could be applied to different substrate ranges. In thepresent disclosure, the organophosphorus degrading enzyme used is theorganophosphorus degrading enzyme of pseudomonas agrobacterium,flavobacterium, or radioactive agrobacterium, thereby enabling thecatalyst to exhibit a higher catalytic efficiency, and to be applied toa wider substrate range.

Pseudomonas agrobacterium, flavobacterium and radioactive agrobacteriumin the present disclosure are commercially available, or obtained by aisolation in the laboratory, and the gene sequences that code theorganophosphate degrading enzyme of the above three kinds of bacteriacould be found from the website (https://www.ncbi.nlm.nih.gov/, amongwhich, pseudomonas agrobacterium is named OPD, GenBank: AER10490.1;flavobacterium is named OPD, GenBank: AAV39527.1; radioactiveagrobacterium is named OPDA, GenBank: AAK85308.1). An affinity tag isintroduced into a gene sequence that codes the organophosphorusdegrading enzyme, which is obtained from one of the above threebacteria, to obtain a desired gene sequence; the desired gene sequenceis transformed in a plasmid; the plasmid is transferred into anexpressing bacteria, i.e., the host bacteria (e.g. escherichia coli) forexpression, and the result of the expression is to obtain a product,i.e. the organophosphorus degrading enzyme required in the presentdisclosure.

The recombinant strain (i.e. escherichia coli above) which can expressthe affinity tag is subjected to a fermentation first, after thefermentation, the cells in the fermentation broth are lyzed, thencentrifuged, to obtain the desired crude enzyme solution oforganophosphorus degrading enzyme with an affinity tag.

According to the present disclosure, after adding the support into thecrude enzyme solution, a mixing is needed to achieve a full binding ofthe support and the target enzyme. The mixing may be performed by usinga shaking table or stirring, preferably by using a shaking table, or byusing other means for mixing known to those skilled in the art. Forusing the shaking table or stirring, in general, the means for mixingcould be selected according to the performance of the support. Forexample, for the support with smaller structural strength, the mixingpreferably is performed by using a shaking table, to avoid thedestruction of the support during the mixing, and for the support withhigh structural strength, the mixing preferably is performed bystirring.

The binding of the support and the target enzyme take a certain periodof time, and the mixing time of the support and target enzyme couldaffect the binding degree of the support and the target enzyme.Generally speaking, within a certain period of time, a longer mixingtime would bring a better binding of the support and the target enzyme.The mixing time in the present disclosure is specifically 0.5-6 h, andmay be, for example, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h or 6 h, preferably 3h. Moreover, in the present disclosure, a better binding of the supportand the target enzyme could be achieved by further optimizing andadjusting the mixing time, in order to improve the enzyme utilizationrate.

According to the present disclosure, after mixing the support and thetarget enzyme to achieve a full binding, the resulting mixture isfurther subjected to a separation to remove fermentation broth, toobtain the organophosphorus degrading enzyme based multifunctionalcatalyst. The separation may be performed by a centrifugation orfiltration, or other means for separation known to those skilled in theart.

The method of the present disclosure is simple in operation, and has alow requirement for the purity of enzyme, and there is no need to carryout costly separation and purification process of the enzyme. Moreover,in the method, the support could be directionally binded with theenzyme, avoiding the destruction of enzyme active site during thebinding. Thus the method enables a high enzyme utilization rate,overcomes the deficiencies of low enzyme utilization rate and highpurity requirement for enzyme during the existing biocatalystpreparation, and could be used to effectively degrade organophosphoruspesticide.

In addition, the present disclosure also involves the organophosphorusdegrading enzyme based multifunctional catalyst prepared by the methodas described above, and use thereof in the degradation oforganophosphorus pesticide.

The organophosphorus degrading enzyme based multifunctional catalystprepared in the present disclosure is low in cost and exhibits highenzyme activity, and thus it is an organophosphorus biologicalnanocatalyst that could be used to effectively degrade organophosphoruspesticide.

The method of the present disclosure will be further illustrated withreference to specific preparation example below.

During the preparation of the organophosphorus degrading enzyme basedmultifunctional catalyst, the composite yolk-shell-structurednanomaterial was used as a support, the gene sequence that codes theorganophosphorus degrading enzyme with an affinity tag was obtained fromradioactive agrobacterium, and escherichia coli was used as expressingbacterium. Thereby, the organophosphorus degrading enzyme labeled withthe affinity tag was prepared.

The specific preparation steps were as follows:

20 mL of 2-methyl imidazole solution (with a concentration of 0.275g/mL) and 3 mL Co(NO₃)₂.6H₂O (with a concentration of 0.15 g/mL)solution were mixed. and sonicated for 10 min, and then stirred at roomtemperature for 6 h, and the resulting mixture was subjected to aseparation by a centrifugation, to obtain a precipitate. The precipitatewas washed in sequence with ultrapure water three times, and with ethylalcohol three times, obtaining a washed precipitate. 0.2 g of the washedprecipitate was dispersed into 60 mL of absolute ethyl alcohol again,and 64 mL of 2-methyl imidazole solution (with a concentration of 0.0625g/mL) was added thereto, and they were uniformly mixed and sonicated for5 min. 4 mL of cetyl trimethyl ammonium chloride (with a concentrationof 5 wt %) solution was added therein, and they were stirred at roomtemperature for 5 min. 1.5 mL of tetraethoxysilane (TEOS) was slowlyadded dropwise therein, and they were stirred at room temperature for1.5 h. The resulting mixture was centrifuged, obtaining a precipitate.The precipitate was washed with ultrapure water and ethyl alcohol threetimes each, obtaining ZIF67@SiO₂.

0.1 g of ZIF67@SiO₂ was uniformly dispersed into 40 mL of ethyl alcoholaqueous solution (5:3) and sonicated for 10 min, and 5 mL of trissolution (with a concentration of 0.04 g/mL) was added into therein,they were mixed. Then 30 mg of dopamine and 75.2 mg of NiCl₂.6H₂O wereadded therein in sequence, and they were stirred at room temperature for15 h after the dopamine and NiCl₂.6H₂O being dissolved completely. Theresulting mixture was centrifuged (9000 rpm, 5 min), obtaining aprecipitate. Finally, the precipitate was washed in sequence withultrapure water three times, and with ethyl alcohol three times,obtaining a precipitate. The precipitate was placed in the oven anddried at 60° C. for 12 h, obtaining a precursor material. The precursormaterial was placed at the center of a quartz crucible and calcined at500° C. for 12 h, with a heating rate of 2° C.·min⁻¹, under theprotection of nitrogen, obtaining black composite yolk-shell-structurednanomaterial

Co/C@SiO₂@Ni/C. The scanning electron micrograph and transmissionelectron micrograph of Co/C@SiO₂@Ni/C were shown in FIG. 1 and FIG. 2.

An affinity tag was introduced into the gene sequence that codes theorganophosphorus degrading enzyme, to obtain a desired gene sequence.The desired gene sequence was transformed in a plasmid, and then theplasmid was transferred into a host escherichia coli. The hostescherichia coli was subjected to fermentation. The cells in thefermentation broth were lyzed after the fermentation, and the resultingmixture was centrifuged, obtaining a crude enzyme solution oforganophosphorus degrading enzyme labeled with the affinity tag.

A certain amount of composite yolk-shell-structured nanomaterial wasadded into a Tris-HCl buffer solution (50 mM, pH 8.0), and they weresonicated for 2 mM to be uniformly dispersed. The uniformly dispersedsolution was added into a crude enzyme solution, and they were uniformlymixed, shook on a shaking table at room temperature for 3 h. Theresulting mixture was subjected to a centrifugation, obtaining aprecipitate. The precipitate was washed with imidazole buffer solution(40 mM) three times, obtaining a washed precipitate, i.e. theorganophosphorus degrading enzyme based multifunctional catalystOpdA@Co/Ni@Ni/C. The specific preparation example could refer to Table 1below.

In order to verify the properties of organophosphorus degrading enzymecatalyst prepared by the present disclosure, to show its ability todegrade organophosphorus pesticide, the inventor used existingorganophosphorus degrading enzyme as a comparative example which waspurchased on the market. The purchased existing organophosphorusdegrading enzyme was a commodity with a brand name of organophosphorusdegrading enzyme biochemical detergent (Model PG-OPH-D1) from TianjinZhangda Science and Technology Development Co., Ltd, China, maincomponent of which was organophosphorus degrading enzyme.

9 mg of the organophosphorus degrading enzyme based multifunctionalcatalysts prepared in Examples 1 to 4 and Comparative product wereweighed respectively, and dispersed into 1.97 mL of Tris-HCl buffersolution (50 mM, pH 9.0), and 20 mg of NaBH₄ and 30 μL ofparathion-methyl (10 mg/mL) were added thereto for reacting for 10 mM at40° C., obtaining a reaction mixture. The reaction mixture wascentrifuged, obtaining a liquid supernatant. 1 mL of the liquidsupernatant was taken and mixed with 1 mL of phenol solution (5% (w/w))and 1 mL of NaOH solution (0.5% (w/w)), obtaining a mixture. The mixturewas subjected to a reaction in a water bath at 30° C. for 30 mM,obtaining a product, i.e. p-aminophenol. The content of p-aminophenolwas measured at 630 nm with a spectrophotometer, and the test resultswere shown in Table 1.

TABLE 1 Source of the organophosphorus Preparation support for degradingenzyme Affinity Content of p- Examples immobilizing gene sequence tagaminophenol Example 1 Composite Radioactive Histidine 0.57 μmolyolk-shell- agrobacterium tag structured nanomaterial Example 2Composite Flavobacterium Histidine 0.46 μmol yolk-shell- tag structurednanomaterial Example 3 Composite Radioactive tryptophan 0.35 μmolyolk-shell- agrobacterium tag structured nanomaterial Example 4Composite Pseudomonas cysteine 0.32 μmol yolk-shell- agrobacterium tagstructured nanomaterial Comparative Existing organophosphorus degrading0.21 μmol Example enzyme

Organophosphorus degrading enzymes could be used to catalyze thedegradation of organophosphorus pesticides into p-nitrophenol. Comparedwith organophosphorus pesticides, p-nitrophenol is much less toxic, butit still has relative high toxicity and still causes pollution to theenvironment. In the presence of hydrogen donor, the compositeyolk-shell-structured nanomaterial could be used to catalyzep-nitrophenol into p-aminophenol, which is a common pharmaceuticalintermediate and has wide application value.

Therefore, it can be seen from the detection results in Table 1 that theorganophosphorus degrading enzyme based multifunctional catalystprepared by the method of the present disclosure has a betterdegradation effect for the organophosphorus pesticide, and exhibits ahigher enzyme utilization rate, thereby having a better degradationeffect, which is conducive to the application in the degradation oforganophosphorus pesticides.

According to the present disclosure, the composite yolk-shell-structurednanomaterial could be used to purify and immobilize the organophosphorusdegrading enzyme with an affinity tag to form the organophosphorusdegrading enzyme based multifunctional catalyst. The prepared catalystcould be used to not only completely degrade organophosphorus pesticide,but also to detect organophosphorus pesticide.

Specifically speaking, a certain amount of the organophosphorusdegrading enzyme based multifunctional catalyst was weighed, and mixedthoroughly with 5 μL of 10 mg/mL parathion-methyl (acetonitrile assolvent) and 995 μL of Tris-HCl buffer (50 mM, pH 8.0), and theresulting mixture was placed in a water bath at 37° C. and incubated for5 min. Then, 1 mL of 10% trichloroacetic acid solution was added thereinto terminate the incubating, and 1 mL of 10% Na₂CO₃ solution was addedthereto for color development. The absorbance value was measured atOD410. Since p-nitrophenol exhibits a characteristic absorption peak at410 nm and appears yellow in the presence of Na₂CO₃, the amount of themethylparathion pesticide could be judged by the color depth.

The above description is only a preferred embodiment of the presentdisclosure, and the present disclosure cannot be limited thereto, andany modifications, equivalent replacements and improvements made withinthe spirit and principle of the present disclosure, should fall withinthe scope of the present disclosure.

1. A method for preparing an organophosphorus degrading enzyme basedmultifunctional catalyst, comprising directly adding a compositeyolk-shell-structured nanomaterial into a crude enzyme solution of anorganophosphorus degrading enzyme with an affinity tag, and mixing, toobtain a mixture, and subjecting the mixture to a separation, to obtainthe organophosphorus degrading enzyme based multifunctional catalyst. 2.The method for preparing the organophosphorus degrading enzyme basedmultifunctional catalyst as claimed in claim 1, wherein the compositeyolk-shell-structured nanomaterial is Co/C@SiO₂@Ni/C.
 3. The method forpreparing the organophosphorus degrading enzyme based multifunctionalcatalyst as claimed in claim 1, wherein the affinity tag is selectedfrom the group consisting of histidine tag, cysteine tag, and tryptophantag.
 4. The method for preparing the organophosphorus degrading enzymebased multifunctional catalyst as claimed in claim 1, wherein theorganophosphorus degrading enzyme is encoded by a gene sequence obtainedfrom one of soil pseudomonas, flavobacterium, and agrobacteriumradioactive.
 5. The method for preparing the organophosphorus degradingenzyme based multifunctional catalyst as claimed in claim 1, wherein themixing is performed by using a shaking table or stirring for 0.5-6hours; subjecting the mixture to a separation is performed by acentrifugation or filtration.
 6. An organophosphorus degrading enzymebased multifunctional catalyst, which is prepared by the method forpreparing the organophosphorus degrading enzyme based multifunctionalcatalyst as claimed in claim
 1. 7. Use of the organophosphorus degradingenzyme based multifunctional catalyst as claimed in claim 6 fordetecting or degrading an organophosphorus pesticide.
 8. The use asclaimed in claim 7, wherein detecting the organophosphorus pesticide isperformed by an optical detection method.
 9. The use as claimed in claim7, wherein the organophosphorus degrading enzyme based multifunctionalcatalyst is used in accompany with a hydrogen donor to degrade theorganophosphorus pesticide.
 10. The use as claimed in claim 9, whereinthe hydrogen donor is sodium borohydride.
 11. The method for preparingthe organophosphorus degrading enzyme based multifunctional catalyst asclaimed in claim 2, wherein the mixing is performed by using a shakingtable or stirring for 0.5-6 hours; subjecting the mixture to aseparation is performed by a centrifugation or filtration.
 12. Themethod for preparing the organophosphorus degrading enzyme basedmultifunctional catalyst as claimed in claim 3, wherein the mixing isperformed by using a shaking table or stirring for 0.5-6 hours;subjecting the mixture to a separation is performed by a centrifugationor filtration.
 13. The method for preparing the organophosphorusdegrading enzyme based multifunctional catalyst as claimed in claim 4,wherein the mixing is performed by using a shaking table or stirring for0.5-6 hours; subjecting the mixture to a separation is performed by acentrifugation or filtration.